Abstract The long term objective of this grant proposal is to elucidate the regulation of sealing ring formation and bone resorption by osteoclasts. During the funding period from July 2004-June 2008, significant progress has been made in understanding the cell biology of cytoskeletal organization associated with bone resorption. Outcome of these studies form the basis for the current application. 1) We have discovered multiplicity of roles for actin-binding and signaling proteins involved in the regulation of sealing ring formation in osteoclasts subjected to bone resorption for 18h, through two dimensional gel electrophoresis and mass spectrometry (MS) analyses. 2) MS analysis in these osteoclasts demonstrated a significant increase in cortactin and corresponding decrease in L-plastin protein levels. 3) Experiments with osteoclasts from L-plastin null mice have shown that although these cells are capable of forming podosomes, they are defective in sealing ring formation and bone resorption. 4) Time- dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. We propose in this application that the secondary actin aggregates formed by L-plastin function as a platform from which actin polymerization commences. This occurs by signaling complex formation through activation of integrin [unreadable]v[unreadable]3-mediated signaling. Src, ERK, WASP, Arp2/3, and cortactin are involved in the regulation of maturation of these actin aggregates to sealing ring during bone resorption. Therefore, our overall hypothesis is that "L-plastin mediates the formation of actin aggregates that act as a precursor to mature sealing rings. Cortactin acts in concert with WASP in the maturation of actin aggregates to form sealing ring by activating Arp2/3 complex-mediated actin polymerization". Thus, our specific aims are: 1) to determine the role of L-plastin in the formation of secondary actin aggregates prior to the formation of sealing ring;2) to elucidate the role of L-plastin phosphorylation in the localization of integrins at the secondary actin aggregates;3) to elucidate the role of cortactin in general and protein-interacting domains of cortactin in particular, in the formation of sealing ring. Different biochemical, cellular, and molecular approaches will be used to advance the understanding of the mechanisms of bone resorption. L-plastin construct will be utilized in an attempt to rescue sealing ring formation in osteoclasts isolated from L-plastin knock-out mice. Identifying the key functional protein(s) and/or reducing bone resorbing activity by small peptides have the potential for pharmacological manipulation. This project should provide fundamental insights into the mechanisms of sealing ring formation and bone resorption. These studies have the potential to provide leads for pharmacological control of osteoporosis, osteoarthritis, and periodontal disease.